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A Bifunctional Polyphosphate Kinase Driving the Regeneration of Nucleoside Triphosphate and Reconstituted Cell-Free Protein Synthesis.

ACS Synth Biol. 2020; 
Wang PH, Fujishima K,, Berhanu S, Kuruma Y,, Jia TZ,, Khusnutdinova AN, Yakunin AF,, McGlynn SE,.
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Gene Synthesis … Corp (Kashiwa, Chiba, Japan) The pETDuet-1 plasmid carrying the recombinant super folder green fluorescent protein (sfGFP) gene (RCSB-PDB ID: 2B3P) was purchased from GenScript (Nanjing, China) The DNA sequences … Get A Quote

摘要

Reconstituted cell-free protein synthesis systems (e.g., the PURE system) allow the expression of toxic proteins, hetero-oligomeric protein subunits, and proteins with noncanonical amino acids with high levels of homogeneity. In these systems, an artificial ATP/GTP regeneration system is required to drive protein synthesis, which is accomplished using three kinases and phosphocreatine. Here, we demonstrate the replacement of these three kinases with one bifunctional Cytophaga hutchinsonii polyphosphate kinase that phosphorylates nucleosides in an exchange reaction from polyphosphate. The optimized single-kinase system produced a final sfGFP concentration (∼530 μg/mL) beyond that of the three-kinase system (... More

关键词

NTP regeneration; family II polyphosphate kinase; functional protein expression; reconstituted cell-free protein synthesis
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